Prostaglandin E(2) inhibits collagen expression and proliferation in patient-derived normal lung fibroblasts via E prostanoid 2 receptor and cAMP signaling

Uncontrolled fibroblast activation is one of the hallmarks of fibrotic lung disease. Prostaglandin E(2) (PGE(2)) has been shown to inhibit fibroblast migration, proliferation, collagen deposition, and myofibroblast differentiation in the lung. Understanding the mechanisms for these effects may provide insight into the pathogenesis of fibrotic lung disease. Previous work has focused on commercially available fibroblast cell lines derived from tissue whose precise origin and histopathology are often unknown. Here, we sought to define the mechanism of PGE(2) inhibition in patient-derived fibroblasts from peripheral lung verified to be histologically normal. Fibroblasts were grown from explants of resected lung, and proliferation and collagen I expression was determined following treatment with PGE(2) or modulators of its receptors and downstream signaling components. PGE(2) inhibited fibroblast proliferation by 33% and collagen I expression by 62%. PGE(2) resulted in a 15-fold increase in intracellular cAMP; other cAMP-elevating agents inhibited collagen I in a manner similar to PGE(2). These effects were reproduced by butaprost, a PGE(2) analog selective for the cAMP-coupled E prostanoid (EP) 2 receptor, but not by selective EP3 or EP4 agonists. Fibroblasts expressed both major cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP-1 (Epac-1), but only a selective PKA agonist was able to appreciably inhibit collagen I expression. Treatment with okadaic acid, a phosphatase inhibitor, potentiated the effects of PGE(2). Our data indicate that PGE(2) inhibits fibroblast activation in primary lung fibroblasts via binding of EP2 receptor and production of cAMP; inhibition of collagen I proceeds via activation of PKA.     

Distinct protein kinase A anchoring proteins direct prostaglandin E2 modulation of Toll-like receptor signaling in alveolar macrophages

Toll-like receptors (TLRs) direct a proinflammatory program in macrophages. One mediator whose generation is induced by TLR ligation is prostaglandin E(2) (PGE(2)), which is well known to increase intracellular cAMP upon G protein-coupled receptor ligation. How PGE(2)/cAMP shapes the nascent TLR response and the mechanisms by which it acts remain poorly understood. Here we explored PGE(2)/cAMP regulation of NO production in primary rat alveolar macrophages stimulated with the TLR4 ligand LPS. Endogenous PGE(2) synthesis accounted for nearly half of the increment in NO production in response to LPS. The enhancing effect of PGE(2) on LPS-stimulated NO was mediated via cAMP, generated mainly upon ligation of the E prostanoid 2 receptor and acting via protein kinase A (PKA) rather than via the exchange protein activated by cAMP. Isoenzyme-selective cAMP agonists and peptide disruptors of protein kinase A anchoring proteins (AKAPs) implicated PKA regulatory subunit type I (RI) interacting with an AKAP in this process. Gene knockdown of potential RI-interacting AKAPs expressed in alveolar macrophages revealed that AKAP10 was required for PGE(2) potentiation of LPS-induced NO synthesis. AKAP10 also mediated PGE(2) potentiation of the expression of cytokines IL-10 and IL-6, whereas PGE(2) suppression of TNF-α was mediated by AKAP8-anchored PKA-RII. Our data illustrate the pleiotropic manner in which G protein-coupled receptor-derived cAMP signaling can influence TLR responses in primary macrophages and suggest that AKAP10 may coordinate increases in gene expression.     

Airway remodeling in murine asthma correlates with a defect in PGE2 synthesis by lung fibroblasts

Asthma is a chronic lung disease characterized by local inflammation that can result in structural alterations termed airway remodeling. One component of airway remodeling involves fibroblast accumulation and activation, resulting in deposition of collagen I around small bronchi. Prostaglandin E(2) (PGE(2)) is the main eicosanoid lipid mediator produced by lung fibroblasts, and it exerts diverse anti-fibrotic actions. Dysregulation of the PGE(2) synthesis/response axis has been identified in human pulmonary fibrotic diseases and implicated in the pathogenesis of animal models of lung parenchymal fibrosis. Here we investigated the relationship between the fibroblast PGE(2) axis and airway fibrosis in an animal model of chronic allergic asthma. Airway fibrosis increased progressively as the number of airway challenges with antigen increased from 3 to 7 to 12. Compared with cells from control lungs, fibroblasts grown from the lungs of asthmatic animals, regardless of challenge number, exhibited no defect in the ability of PGE(2) or its analogs to inhibit cellular proliferation and collagen I expression. This correlated with intact expression of the EP(2) receptor, which is pivotal for PGE(2) responsiveness. However, cytokine-induced upregulation of PGE(2) biosynthesis as well as expression of cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 declined with increasing numbers of antigen challenges. In addition, treatment with the COX-2-selective inhibitor nimesulide potentiated the degree of airway fibrosis following repeated allergen challenge. Because endogenous COX-2-derived PGE(2) acts as a brake on airway fibrosis, the inability of fibroblasts to upregulate PGE(2) generation in the inflammatory milieu presented by repeated allergen exposure could contribute to the airway remodeling and fibrosis observed in chronic asthma.     

Cyclic AMP Signaling Reduces Sirtuin 6 Expression in Non-small Cell Lung Cancer Cells by Promoting Ubiquitin-Proteasomal Degradation via Inhibition of the Raf-MEK-ERK (Raf/Mitogen-activated Extracellular Signal-regulated Kinase/Extracellular Signal-regulated Kinase) Pathway

The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gα_s plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.     

Tissue-specific mechanisms for CCN2/CTGF persistence in fibrotic gingiva: interactions between cAMP and MAPK signaling pathways, and prostaglandin E2-EP3 receptor mediated activation of the c-JUN N-terminal kinase

Prostaglandin E(2) blocks transforming growth factor TGF beta1-induced CCN2/CTGF expression in lung and kidney fibroblasts. PGE(2) levels are high in gingival tissues yet CCN2/CTGF expression is elevated in fibrotic gingival overgrowth. Gingival fibroblast expression of CCN2/CTGF in the presence of PGE(2) led us to compare the regulation of CCN2/CTGF expression in fibroblasts cultured from different tissues. Data demonstrate that the TGFbeta1-induced expression of CCN2/CTGF in human lung and renal mesangial cells is inhibited by 10 nm PGE(2), whereas human gingival fibroblasts are resistant. Ten nm PGE(2) increases cAMP accumulation in lung but not gingival fibroblasts, which require 1 mum PGE(2) to elevate cAMP. Micromolar PGE(2) only slightly reduces the TGFbeta1-stimulated CCN2/CTGF levels in gingival cells. EP2 prostaglandin receptor activation with butaprost blocks the TGFbeta1-stimulated expression of CCN2/CTGF expression in lung, but not gingival, fibroblasts. In lung fibroblasts, inhibition of the TGFbeta1-stimulated CCN2/CTGF by PGE(2), butaprost, or forskolin is due to p38, ERK, and JNK MAP kinase inhibition that is cAMP-dependent. Inhibition of any two MAPKs completely blocks CCN2/CTGF expression stimulated by TGFbeta1. These data mimic the inhibitory effects of 10 nm PGE(2) and forskolin that were dependent on PKA activity. In gingival fibroblasts, the sole MAPK mediating the TGFbeta1-stimulated CCN2/CTGF expression is JNK. Whereas forskolin reduces TGFbeta1-stimulated expression of CCN2/CTGF by 35% and JNK activation in gingival fibroblasts, micromolar PGE(2)-stimulated JNK in gingival fibroblasts and opposes the inhibitory effects of cAMP on CCN2/CTGF expression. Stimulation of the EP3 receptor with sulprostone results in a robust increase in JNK activation in these cells. Taken together, data identify two mechanisms by which TGFbeta1-stimulated CCN2/CTGF levels in human gingival fibroblasts resist down-regulation by PGE(2): (i) cAMP cross-talk with MAPK pathways is limited in gingival fibroblasts; (ii) PGE(2) activation of the EP3 prostanoid receptor stimulates the activation of JNK.     

Cyclic adenosine 5'-monophosphate-stimulated neurotensin secretion is mediated through Rap1 downstream of both Epac and protein kinase A signaling pathways

Neurotensin (NT), a gut peptide, plays important roles in gastrointestinal secretion, inflammation, and growth of normal and neoplastic tissues. cAMP regulates the secretion of hormones via its effector proteins protein kinase A (PKA) or Epac (exchange protein directly activated by cAMP). The small GTPase Rap1 can be activated by both PKA and Epac; however, the role of Rap1 in hormone secretion is unknown. Here, using the BON human endocrine cell line, we found that forskolin (FSK)-stimulated NT secretion was reduced by inhibition of Rap1 expression and activity. FSK-stimulated NT secretion was enhanced by overexpression of either wild-type or constitutively active Rap1. Epac activators and wild-type Epac enhanced NT release and Rap1 activity. In contrast, overexpression of a cAMP binding mutant, EpacR279E, decreased NT release and Rap1 activity. PKA activation increased NT release and Rap1 activity. FSK-stimulated NT release was reduced by PKA inhibition and the dominant negative Rap1N17. NT secretion, stimulated by Epac activation, was reduced by PKA inhibition; NT release, stimulated by PKA activation, was enhanced by wild-type Epac but reduced by the mutant EpacR279E. Finally, prostaglandin E2 (PGE2), a physiological agent that increases cAMP, stimulated NT secretion via cAMP/PKA/Rap1. Importantly, we demonstrate that PKA and Epac mediate the cAMP-induced NT secretion synergistically by converging at the common downstream target protein Rap1. Moreover, PGE2, a potent mediator of inflammation and associated with colorectal carcinogenesis, stimulates NT release suggesting a possible link between PGE2 and NT on intestinal inflammatory disorders and colorectal cancers.     

Bleomycin-induced E prostanoid receptor changes alter fibroblast responses to prostaglandin E2

Although PGE(2) is a potent inhibitor of fibroblast function, PGE(2) levels are paradoxically elevated in murine lungs undergoing fibrotic responses. Pulmonary fibroblasts from untreated mice expressed all four E prostanoid (EP) receptors for PGE(2). However, following challenge with the fibrogenic agent, bleomycin, fibroblasts showed loss of EP2 expression. Lack of EP2 expression correlated with an inability of fibroblasts from bleomycin-treated mice to be inhibited by PGE(2) in assays of proliferation or collagen synthesis and blunted cAMP elevations in response to PGE(2). PGE(2) was similarly unable to suppress proliferation or collagen synthesis in fibroblasts from EP2(-/-) mice despite expression of the other EP receptors. EP2(-/-), but not EP1(-/-) or EP3(-/-) mice, showed exaggerated fibrotic responses to bleomycin administration in vivo as compared with wild-type controls. EP2 loss on fibroblasts was verified in a second model of pulmonary fibrosis using FITC. Our results for the first time link EP2 receptor loss on fibroblasts following fibrotic lung injury to altered suppression by PGE(2) and thus identify a novel fibrogenic mechanism.     

Novel mechanism of cyclic AMP mediated extracellular signal regulated kinase activation in an intestinal cell line

The extracellular signal regulated kinase (ERK1/2) signaling cascade has been implicated as both a pro-apoptotic and anti-apoptotic pathway depending on cell type and context. In the T84 intestinal epithelial cell line, cAMP activates ERK1/2 resulting in the inhibition of apoptosis. Cyclic-AMP signaling relies on the binding and activation of a cAMP binding protein. In most cell types, the majority of this signaling occurs through an isoform of protein kinase A (PKAI or PKAII). Despite evidence to the contrary, we hypothesized that ERK1/2 activation is through a PKA isoform. Pharmacological activators and inhibitors of PKA as well as siRNA were used to further interrogate this potential signaling pathway. Our results demonstrate that at doses sufficient to increase PKA activity, PKAII specific cAMP analogs activate ERK1/2 while PKAI analogs do not. Pharmacological inhibition of the PKAII regulatory subunit and catalytic subunit as well as siRNA knockdown of the catalytic subunit blocks ERK1/2 activation. We conclude that in the T84 cell line, cAMP binding to the PKAII regulatory subunit leads to the subsequent phosphorylation of ERK1/2 and provides insight into the mechanism of cAMP mediated survival signaling in the intestinal epithelium. These results directly implicate PKAII as a mediator of cell survival in T84 cells and provide evidence for an additional means by which cAMP can influence intestinal cell turnover.     

Signaling pathways involving adenosine A<Subscript>2A</Subscript> and A<Subscript>2B</Subscript> receptors in wound healing and fibrosis

Collagen and matrix deposition by fibroblasts is an essential part of wound healing but also contributes to pathologic remodeling of organs leading to substantial morbidity and mortality. Adenosine, a small molecule generated extracellularly from adenine nucleotides as a result of direct stimulation, hypoxia, or injury, acts via a family of classical seven-pass G protein-coupled protein receptors, A_2A and A_2B, leading to generation of cAMP and activation of downstream targets such as PKA and Epac. These effectors, in turn, lead to fibroblast activation and collagen synthesis. The regulatory actions of these receptors likely involve multiple interconnected pathways, and one of the more interesting aspects of this regulation is opposing effects at different levels of cAMP generated. Additionally, adenosine signaling contributes to fibrosis in organ-specific ways and may have opposite effects in different organs. The development of drugs that selectively target these receptors and their signaling pathways will disrupt the pathogenesis of fibrosis and slow or arrest the progression of the important diseases they underlie.     

Inhibition of phospholipase A2-mediated arachidonic acid release by cyclic AMP defines a negative feedback loop for P2Y receptor activation in Madin-Darby canine kidney D1 cells

In Madin-Darby canine kidney D1 cells extracellular nucleotides activate P2Y receptors that couple to several signal transduction pathways, including stimulation of multiple phospholipases and adenylyl cyclase. For one class of P2Y receptors, P2Y2 receptors, this stimulation of adenylyl cyclase and increase in cAMP occurs via the conversion of phospholipase A2 (PLA2)-generated arachidonic acid (AA) to prostaglandins (e.g. PGE2). These prostaglandins then stimulate adenylyl cyclase activity, presumably via activation of prostanoid receptors. In the current study we show that agents that increase cellular cAMP levels (including PGE2, forskolin, and the beta-adrenergic agonist isoproterenol) can inhibit P2Y receptor-promoted AA release. The protein kinase A (PKA) inhibitor H89 blocks this effect, suggesting that this feedback inhibition occurs via activation of PKA. Studies with PGE2 indicate that inhibition of AA release is attributable to inhibition of mitogen-activated protein kinase activity and in turn of P2Y receptor stimulated PLA2 activity. Although cAMP/PKA-mediated inhibition occurs for P2Y receptor-promoted AA release, we did not find such inhibition for epinephrine (alpha1-adrenergic) or bradykinin-mediated AA release. Taken together, these results indicate that negative feedback regulation via cAMP/PKA-mediated inhibition of mitogen-activated protein kinase occurs for some, but not all, classes of receptors that promote PLA2 activation and AA release. We speculate that receptor-selective feedback inhibition occurs because PLA2 activation by different receptors in Madin-Darby canine kidney D1 cells involves the utilization of different signaling components that are differentially sensitive to increases in cAMP or, alternatively, because of compartmentation of signaling components.     

Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.     

Antifibrotic Effects of Noscapine through Activation of Prostaglandin E2 Receptors and Protein Kinase A

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-β-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-β-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP₂ prostaglandin E₂ receptor antagonist, PF-04418948, but not by the antagonists of EP₄, prostaglandin D₂, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP₂ prostaglandin E₂ receptor-mediated activation of PKA in pulmonary fibroblasts.     

Extracellular calcium induces COX-2 in osteoblasts via a PKA pathway

We have shown that extracellular calcium [Ca(+2)](e) induces cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) production via an ERK signaling pathway in osteoblasts. In this study, we examined the roles of protein kinase C (PKC) and A (PKA) signaling pathways in the [Ca(+2)](e) induction of COX-2 in primary calvarial osteoblasts from mice transgenic for -371 bp of the COX-2 promoter fused to a luciferase reporter. Neither PKC specific inhibitors nor downregulation of the PKC pathway by phorbol myristate acetate (PMA) affected the [Ca(+2)](e) stimulation of COX-2 mRNA or promoter activity. In contrast, PKA inhibitors, used at doses that inhibited forskolin-stimulated luciferase activity by 90%, reduced [Ca(+2)](e)-stimulated COX-2 mRNA expression and promoter activity by 80-90%. [Ca(+2)](e) also stimulated a 2- to 3-fold increase in cAMP production. Hence, the [Ca(+2)](e) induction of COX-2 mRNA expression and promoter activity was independent of the PKC pathway and dependent on the PKA signaling pathway.     

Matrix stiffness controls cardiac fibroblast activation through regulating YAP via AT1R

Cardiac fibrosis is a common pathway leading to heart failure and involves continued activation of cardiac fibroblasts (CFs) into myofibroblasts during myocardium damage, causing excessive deposition of the extracellular matrix (ECM) and thus increases matrix stiffness. Increasing evidence has shown that stiffened matrix plays an important role in promoting CF activation and cardiac fibrosis, and several signaling factors mediating CF mechanotransduction have been identified. However, the key molecules that perceive matrix stiffness to regulate CF activation remain to be further explored. Here, we detected significantly increased expression and nuclear localization of Yes-associated protein (YAP) in native fibrotic cardiac tissues. By using mechanically regulated in vitro cell culture models, we found that a stiff matrix-induced high expression and nuclear localization of YAP in CFs, accompanied by enhanced cell activation. We also demonstrated that YAP knockdown decreased fibrogenic response of CFs and that YAP overexpression promoted CF activation, indicating that YAP plays an important role in mediating matrix stiffness-induced CF activation. Further mechanistic studies revealed that the YAP pathway is an important signaling branch downstream of angiotensin II type 1 receptor in CF mechanotransduction. The findings help elucidate the mechanism of fibrotic mechanotransduction and may contribute to the development of new approaches for treating fibrotic diseases.     

Prostaglandin E2 protects gastric mucosal cells from apoptosis via EP2 and EP4 receptor activation

Prostaglandin E(2) (PGE(2)) has a strong protective effect on the gastric mucosa in vivo; however, the molecular mechanism of a direct cytoprotective effect of PGE(2) on gastric mucosal cells has yet to be elucidated. Although we reported previously that PGE(2) inhibited gastric irritant-induced apoptotic DNA fragmentation in primary cultures of guinea pig gastric mucosal cells, we show here that PGE(2) inhibits the ethanol-dependent release of cytochrome c from mitochondria. Of the four main subtypes of PGE(2) receptors, we also demonstrated, using subtype-specific agonists, that EP(2) and EP(4) receptors are involved in the PGE(2)-mediated protection of gastric mucosal cells from ethanol-induced apoptosis. Activation of EP(2) and EP(4) receptors is coupled with an increase in cAMP, for which a cAMP analogue was found here to inhibit the ethanol-induced apoptosis. The increase in cAMP is known to activate both protein kinase A (PKA) and phosphatidylinositol 3-kinase pathways. An inhibitor of PKA but not of phosphatidylinositol 3-kinase blocked the PGE(2)-mediated protection of cells from ethanol-induced apoptosis, suggesting that a PKA pathway is mainly responsible for the PGE(2)-mediated inhibition of apoptosis. Based on these results, we considered that PGE(2) inhibited gastric irritant-induced apoptosis in gastric mucosal cells via induction of an increase in cAMP and activation of PKA, and that this effect was involved in the PGE(2)-mediated protection of the gastric mucosa from gastric irritants in vivo.